Inhibitors of β-lactamases and uses therefor

ABSTRACT

The invention provides novel non-β-lactam inhibitors of β-lactamases. In particular, the invention provides such inhibitors which are boronic acids of formula (1) which is set forth in the specification. These compounds may be used with β-lactam antibiotics to treat β-lactam-antibiotic-resistant bacterial infections. These compounds are also antibacterial by themselves. Finally, the invention provides a pharmaceutical composition comprising these compounds.

Benefit of provisional application No. 60/049,992, filed Jun. 13, 1997,is hereby claimed.

BACKGROUND

Bacterial resistance to antibiotics has raised fears of an approachingmedical catastrophe (Neu, Science, 257, 1064-1073 (1992)). Evolutionaryselection and genetic transformation have made this problem pressing.Most antibiotic drugs are derivatives of naturally occurringbactericides (Davies, Science, 264, 375-382 (1994)), and many resistancemechanisms evolved long ago. Human use of antibiotics has refined thesemechanisms and promoted their spread through gene transfer (Davies,Science, 264, 375-382 (1994)). A resistance mechanism originating in onespecies of bacteria can be expected to spread throughout the biosphere.

Bacterial adaptations to β-lactam drugs (e.g., amoxicillin, cephalothin,clavulanate, aztreonam) are among the best studied and most perniciousforms of antibiotic resistance. β-lactams target enzymes that are uniqueto bacteria and are thus highly selective. They have been widelyprescribed. In the absence of resistance, β-lactams are the first choicefor treatment in 45 of 78 common bacterial infections (Goodman &Gilman's The Pharmacological Basis of Therapeutics (Hardman et al.,eds., McGraw-Hill, New York, 1996)). The evolution of resistance tothese drugs has raised the cost of antibiotic therapy and reduced itseffectiveness, leading to increased rates of morbidity and mortality.

β-lactam antibiotics inhibit bacterial cell wall biosynthesis (Tomasz,Rev. Infect. Dis., 8, S270-S278 (1986)). The drugs form covalentcomplexes with a group of transpeptidases/carboxypeptidases calledpenicillin binding proteins (PBPs). PBP inactivation disrupts cell wallbiosynthesis, leading to self-lysis and death of the bacteria.

Bacteria use several different mechanisms to escape from β-lactam drugs(Sanders, Clinical Infectious Disease, 14, 1089-1099 (1992); Li et al.,Antimicrob. Agents Chemother., 39, 1948-1953 (1995)). Probably the mostwidespread is the hydrolysis of β-lactams by β-lactamase enzymes.

TEM-1 and AmpC are two β-lactamases from Escherichia coli. E. coli is animportant pathogen in its own right. It is the most common cause ofgram-negative bacterial infection in humans (Levine, New Engl. J. Med.,313, 445-447 (1985)), and is the most prevalent hospital-acquiredinfection (Thornsberry, Pharmacotherapy, 15, S3-8 (1995)). E. coli thatcarry TEM-1, or for which AmpC production has been derepressed, areresistant to β-lactam treatment. As of 1992, as many of 30% ofcommunity-isolated E. coli and 40-50% of hospital-acquired E. coli inthe United States were resistant to β-lactams such as amoxicillin (Neu,Science, 257, 1064-1073 (1992)). Many of these resistant E. coli areresistant to β-lactamase inhibitors such as clavulanic acid andsulbactam.

TEM-1 and AmpC are major forms of plasmid-based and chromosomalβ-lactamases and are responsible for resistance in a broad host range.The versions of TEM and AmpC (Galleni, et al., Biochem. J., 250, 753-760(1988)) in other bacterial species share high sequence identity to TEM-1and AmpC from E. coli. TEM-1 structurally and catalytically resemblesthe class A β-lactamase from Staphlococcus aureus. The structures ofAmpC from Citrobacter freundii and Enterobacter cloacae have beendetermined, and they closely resemble the structure of the E. colienzyme (K. Usher, L. Blaszczak, B. K. Shoichet, J. R. Remington, inpreparation (1996)).

To overcome the action of β-lactamases, medicinal chemists haveintroduced compounds that inhibit these enzymes, such as clavulanicacid, or compounds that are less susceptible to enzyme hydrolysis, suchas aztreonam. Both have been widely used in antibiotic therapy(Rolinson, Rev. Infect. Diseases 13, S727-732 (1991)); both areβ-lactams. Their similarity to the drugs that they are meant to protector replace has allowed bacteria to evolve further, maintaining theirresistance.

Resistance to these new classes of β-lactams has arisen throughmodifications of previously successful mechanisms. Point substitutionsin β-lactamases allow the enzymes to hydrolyze compounds designed toevade them (Philippon et al., Antimicrob. Agents Chemother., 33,1131-1136 (1989)). Other substitutions reduce the affinity of β-lactaminhibitors for the enzymes (Saves, et al., J. Biol. Chem., 270,18240-18245 (1995)) or allow the enzymes to simply hydrolyze them.Several gram positive bacteria, such as Staph. aureus, have acquiredsensor proteins that detect β-lactams in the environment of the cell(Bennet and Chopra, Antimicrob. Agents Chemotherapy, 37, 153-158(1993)). β-lactam binding to these sensors leads to transcriptionalup-regulation of the β-lactamase. β-lactam inhibitors of β-lactamases,thus, can induce the production of the enzyme that they are meant toinhibit, defeating themselves.

It is noteworthy that the human therapeutic attack on bacteria hasparalleled the path taken in nature. Several species of soil bacteriaand fungi produce β-lactams, presumably as weapons against otherbacteria (although this remains a matter of debate). Over evolutionarytime, susceptible bacteria have responded to β-lactams withβ-lactamases, among other defenses. In turn, soil bacteria have producedβ-lactams that resist hydrolysis by β-lactamases or have producedβ-lactams that inhibit the β-lactamases. Streptomyces clavuligeris makesseveral β-lactams, including clavulanic acid, a clinically usedinhibitor of class A β-lactamases such as TEM-1. Chromobacteriumviolaceum makes aztreonam, a clinically used monobactam that resistshydrolysis by many β-lactamases. One reason why bacteria have been ableto respond rapidly with "new" resistance mechanisms to β-lactams, andindeed many classes of antibiotics, is that the mechanisms are not infact new. As long as medicinal chemistry focuses on new β-lactammolecules to overcome β-lactamases, resistance can be expected to followshortly. The logic will hold for any family of antibiotic where the leaddrug, and resistance mechanisms to it, originated in the biosphere longbefore their human therapeutic use. This includes the aminoglycosides,chloramphenicol, the tetracyclines and vancomycin.

One way to avoid recapitulating this ancient "arms race" would be todevelop inhibitors that have novel chemistries, dissimilar to β-lactams.These non-β-lactam inhibitors would not themselves be degraded byβ-lactamases, and mutations in the enzymes should not render them labileto hydrolysis. Novel inhibitors would escape detection by β-lactamsensor proteins that up-regulate β-lactamase transcription, and may beunaffected by porin mutations that limit the access of β-lactams toPBPs. Such inhibitors would allow current β-lactam drugs to work againstbacteria where β-lactamases provide the dominant resistance mechansim.

It has previously been reported that boric acid and certain phenylboronic acids are inhibitors of certain β-lactamases. See, Kiener andWaley, Biochem. J., 169, 197-204 (1978) (boric acid, phenylboronic acid(2FDB) and m-aminophenylboronate (MAPB)); Beesley et al., Biochem. J.,209, 229-233 (1983) (twelve substituted phenylborinic acids, including2-formylphenylboronate (2FORMB), 4-formylphenylboronate (4FORMB), and4-methylphenylboronate (4MEPB)); Amicosante et al., J. Chemotherapy, 1,394-398 (1989) (boric acid, 2FDB, MAPB and tetraphenylboronic acid).More recently, m-(dansylamidophenyl)-boronic acid (NSULFB) has beenreported to be a submicromolar inhibitor of the Enterobacter cloacae P99β-lactamase. Dryjanski and Pratt, Biochemistry, 34, 3561-3568 (1995). Inaddition, Strynadka and colleagues used the crystallographic structureof a mutant TEM-1 enzyme-penicillin G complex to design a novelalkylboronic acid inhibitor [(1R)-1-acetamido-2-(3-carboxyphenyl)ethaneboronic acid] with high affinity for this enzyme. Strynadka et al., Nat.Struc. Biol., 3, 688-695 (1996).

SUMMARY OF THE INVENTION

The invention provides non-β-lactam inhibitors of β-lactamases. Inparticular, the invention provides β-lactamase inhibitors having theformula: ##STR1## wherein: R is naphthalene, phenanthrene, or has one ofthe following formulas: ##STR2## wherein: ring system (2), (3), (4),(5), (6), (7), (8), (9) or (10) is aromatic or nonaromatic;

the atom center * is (R) or (S) in the case of chiral compounds;

positions 1, 2, 3, 4, 5, 6, 7 or 8 each independently is C, N, O or S;

R₁ through R₆ each independently is a lone pair, H, B(OH)₂, a halogenatom, CF₃, CH₂ CF₃, CCl₃, CH₂ CCl₃, CBr₃, CH₂ CBr₃, NO₂, lower alkyl,CO₂ H, CHCHCOOH, CH₂ CH₂ CH₂ COOH, SO₃ H, PO₃ H, OSO₃ H, OPO₃ H, OH,NH₂, CONH₂, COCH₃, OCH₃, or phenyl boronic acid, except that R₂, R₃, R₄,R₅ and R₆ cannot all simultaneously be H, R₂ cannot be lower alkyl whenR₃, R₄, R₅ and R₆ are H, R₃ cannot be NH₂, OH or lower alkyl when R₂,R₄, R₅ and R₆ are H, and R₄ cannot be lower alkyl when R₂, R₃, R₅ and R₆are H;

R₇ is a lone pair, H, B(OH)₂, a halogen atom, CF₃, CCl₃, CBr₃, CH₂ CF₃,CH₂ CCl₃, CH₂ CBr₃, NO₂, CONH₂, COCH₃, OCH₃, lower alkyl, aryl, arylsubstituted with one or more substituents R₈, heteroaryl, or heteroarylsubstituted with one or more substituents R₈ ;

each R₈ is independently a lone pair, H, B(OH)₂, a halogen atom, CF₃,CCl₃, CBr₃, CH₂ CF₃, CH₂ CCl₃, CH₂ CBr₃, NO₂, lower alkyl, O, N, S, OH,NH₂, N(CH₃)₂, N(CH₃)CH₂ CH₃, NCOCH₃, COOH, CHCHCOOH, CH₂ CH₂ CH₂ COOH,CONH₂, COCH₃, OCH₃, OCl or phenyl boronic acid;

X is O, NH, NCH₃ or ##STR3## Y is OH, NH₂, NCH₃, N(CH₃)₂, NHCOCH₃ orNHCOCH₂ COOH; and

R₉ is a lone pair, H, B(OH)₂, a halogen atom, CF₃, CCl₃, CBr₃, CH₂ CF₃,CH₂ CCl₃, CH₂ CBr₃, NO₂, CO₂ H, CHCHCOOH, CH₂ CH₂ CH₂ COOH, SO₃ H, PO₃H, OSO₃ H, OPO₃ H, OH, NH₂, CONH₂, COCH₃, OCH3, phenyl boronic acid,lower alkyl, or a side chain of a standard amino acid.

The invention also provides a method of treating aβ-lactam-antibiotic-resistant bacterial infection. The method comprisesadministering to an animal suffering from such an infection an effectiveamount of a β-lactamase inhibitor of formula (1), or apharmaceutically-acceptable salt thereof, and an effective amount of aβ-lactam antibiotic.

It has also been found that the compounds of formula (1), andpharmaceutically-acceptable salts thereof, are antibacterial bythemselves. Thus, the invention further provides a method of treating abacterial infection comprising administering to an animal suffering fromsuch an infection an effective amount of a compound of formula (1), or apharmaceutically-acceptable salt thereof.

Finally, the invention provides pharmaceutical compositions comprisingcompounds of formula (1), or pharmaceutically-acceptable salts thereof,and a pharmaceutically-acceptable carrier. The pharmaceuticalcompositions may also comprise β-lactam antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E. Structures of boronic acids. FIG. 1A--boronic acidspreviously reported to be inhibitors of β-lactamase (prior art). FIGS.1B and 1C--crystal structure of the complex of AmpC β-lactamase and theboronic acid inhibitor m-aminophenylboronic acid (MAPB). Note that them-amino group of MAPB is not shown for clarity and that only theside-chains of most of the neighboring residues in the AmpC active siteare shown for the same reason. The positions of the MAPB phenyl ring arenumbered in both the `top` (FIG. 1B) and `back` (FIG. 1C) views. FIGS.1D and 1E--boronic acid inhibitors of β-lactamases, including prior artinhibitors (marked with an *) and inhibitors according to the invention.

FIGS. 2A-C. Diagrams of the synthesis of compounds of formula (1)wherein R is (4).

FIG. 3. Diagram of the synthesis of compounds of formula (1) wherein Ris (12).

FIGS. 4A-B. Diagrams of the synthesis of compounds of formula (1)wherein R is (11).

FIG. 5. Representation of the environment around β-lactamase inhibitorbenzo[b]thiophene-2-boronic acid (BZBTH2B) showing key active siteresidues of the enzyme.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS OF THEINVENTION

In formula (1) above, the following terms have the following meanings.

A "lone pair" refers to an unshared pair of electrons (not involved inan actual covalent chemical bond to another atom) that may haveimportant interactions in receptor-ligand (e.g., enzyme-inhibitor)complexes.

"Alkyl" means a straight- or branched-chain alkyl containing 1-25 carbonatoms. "Lower alkyl" means a straight- or branched-chain alkylcontaining 1-4 carbon atoms. Both of these terms include the R and Sisomers.

"Aryl" means a structure containing from 1 to 3 aromatic rings, eachring containing from 5 to 6 carbon atoms.

"Heteroaryl" means an aryl as defined above wherein the ring(s) containone or more atoms of S, N or O.

The "standard amino acids" are alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,homoserine, hydroxyproline, isoleucine, leucine, lysine, methionine,norleucine, norvaline, ornithine, penicillamine, phenylalanine,phenylglycine, proline, pyroglutamic acid, serine, threonine,tryptophan, tyrosine, and valine. Both the D and L isomers can be used.The side chains of these amino acids are well known and are the portionsof the amino acids attached to the NH₂ --CH--COOH backbone. Forinstance, the side chain of alanine is CH₃ and the side chain ofasparagine is CH₂ CONH₂.

The most preferred compounds of formula (1) are those wherein R is (4).Particularly preferred are those compounds wherein atom 1 of (4) is S orO, most preferably S, and the remaining atoms are carbons. Of this groupof compounds, each R₁ is preferably H or each R₁ is H, except for the R₁'s attached to atoms numbers 3, 4 and 6. Preferably the R₁ attached toatom 6 is lower alkyl and the R₁ 's attached to atoms 3 and 4 are small,polar and capable of forming hydrogen bonds. Most preferably the R₁attached to atom 3 is COOH, CHCHCOOH or CONH₂, and the R₁ attached toatom 4 is NH₂. The most preferred compounds are benzo[b]furan-2-boronicacid and benzo[b]thiophene-2-boronic acid.

Other preferred compounds of formula (1) are those wherein R is (6).Particularly preferred are those compounds wherein atom 2 of (6) is Sand the remaining atoms are carbons. Of this group of compounds, each R₁is preferably H. The most preferred compound isbenzo[b]thiophene-3-boronic acid.

Also preferred are compounds of formula (1) wherein R is (2). When R is(2), atom 1 is preferably S or atom 2 is preferably S or O. Mostpreferably atom 1 is S or atom 2 is O. Especially preferred compoundsare thiophene-2-boronic acid, 3-formylthiophene-2-boronic acid,5-chlorothiophene-2-boronic acid, 4-methylthiophene-2-boronic acid,5-acetylthiophene-2-boronic acid, and R-3-tetrahydrofuranylboronic acid.

Other preferred compounds of formula (1) are those wherein R is (11).When R is (11), X preferably is O, NCH₃ or ##STR4## wherein Y ispreferably NH₂ and R₁₉ is preferably the side chain of a polar, but notcharged, amino acid (e.g., serine, threonine, asparagine and glutamine).Most preferred are 4-(3-boronatophenylazo)homophthalic anhydride and4-(3-boronatophenylazo)-2-methylhomophthalimide.

Further preferred compounds of formula (1) include those wherein R is(3), and in (3) atoms 1-5 are all carbons and the ring is aryl.Preferred substituents R₂ -R₆ include halogen, lower alkyl substitutedwith one or more halogen atoms (e.g., CF₃), NO₂, CHCHCOOH and phenylboronic acid. More preferred are phenylboronic acid and NO₂, with NO₂being the most preferred.

Additional preferred compounds of formula (1) include those wherein R is(12). Preferably R₁ is OH. Preferably R₇ is aryl or heteroaryl,unsubstituted or substituted with one or more substituents R₈. Mostpreferably R₇ is phenyl substituted with one or more substituents R₈.Most preferred are 2-hydroxy-5-(3-trifluoromethylphenylazo)benzeneboronic acid and2,4,6-tris(5-(4-bromophenylazo)-2-hydroxyphenyl)boroxin.

The compounds of formula (1) are available commercially or can besynthesized as described below. Commercial sources of the compoundsinclude TCI America, Portland, Oreg.; Key Organics, Cornwall, UK;Bionet, Cornwall, UK; Frontier Scientific, Logan, Utah; AldrichChemical, Milwaukee, Wis.; and Lancaster Synthesis, Windham, N.H.

Also, unless otherwise noted, the various chemicals used in thesyntheses described below are available from commercial sourcesincluding Aldrich Chemical, Milwaukee, Wis., Lancaster Synthesis,Windham, N.H., TCI America, Portland, Oreg., Sigma Chemical Co., St.Louis, Mo., Acros Organics, Pittsburgh, Pa., Chemservice Inc., WestChester, Pa., BDH Inc., Toronto, Canada, Fluka Chemical Corp.,Ronkonkoma, N.Y., Pfaltz & Bauer, Inc., Waterbury, Conn., AvocadoResearch, Lancashire, UK, Crescent Chemical Co., Hauppauge, N.Y., FisherScientific Co., Pittsburgh, Pa., Fisons Chemicals, Leicestershire, UK,ICN Biomedicals, Inc., Costa Mesa, Calif., Pierce Chemical Co.,Rockford, Ill., Riedel de Haen AG, Hannover, Germany, Wako ChemicalsUSA, Inc., Richmond, Va., Maybridge Chemical Co. Ltd., Cornwall, UK,Trans World Chemicals, Inc., Rockville, Md., Apin Chemicals Ltd., MiltonPark, UK, and Parish Chemical Co., Orem, Utah.

Compounds of formula (1) wherein R is (2)-(10), (13) and (14) can besynthesized as described in Beesley et al., Biochem. J., 209, 229-233(1983) or Matteson, Acc. Chem. Res., 21, 294-300 (1988). Also see FIGS.2A-C which diagram methods of synthesizing compounds of formula (1)wherein R is (4). In these figures, BuLi is butyl lithium. R_(x), R_(y)and R₂ may be any suitable leaving group such as lower alkyl,cycloalkyl, or phenyl. R₁ is defined above.

Compounds of formula (1) wherein R is (12) can be synthesized asdepicted in FIG. 3 using R₇ --N═O as the starting compound. Thepolystyrene resin, P, can be functionalized as described in Leznoff andWong, Can. J. Chem., 51:3756-3764 (1973). Alternatively, functionalizedresins can be purchased from Novabiochem. The reaction (b) in FIG. 3 iscalled the Mills reaction. The selectivity of the hydrolysis in theMills reaction is determined by the temperature, which should be keptlow. Glacial acetic acid is used in this step when R₇ is aryl. However,the acidic conditions must be varied depending on the R₇ group, andother solvents and mineral acids are used. See March, Advanced OrganicChemistry, page 638 (4th ed. 1992) (John Wiley and Sons) and TheChemistry Of Nitro and Nitroso Groups, part 1, pages 278-283 (1969)(Interscience, New York). A modification of this reaction is describedin Ayyangar et al., Tetrahedron Letters, 30, 7253 (1989) (starting with3-N-acylphenyl boronic acid instead of 3-aminophenyl boronic acid).

Finally, FIGS. 4A-B are diagrams of methods of synthesizing compounds offormula (1) wherein R is (11). In FIG. 4A, the reaction is preferablycarried out using the free boronic acid as shown in FIG. 4A. However,functionalized resins can be used, as illustrated in FIG. 3. The use ofsuch resins reduces the risk of secondary reactions due to sterichindrance. However, if the resin is used, the second step (reducing thediazonium salt to the hydrazine) will result in cleavage from the resin.In either case, the boronic acid can provide the acidic conditions forthe reaction. In FIG. 4B, U is H, CH₃ or R₉ CH₂ COY. Compounds offormula (1) wherein R is (11) may also be obtained from Key Organics,Cornwall, U.K. (custom synthesis).

The compounds of formula (1) may contain an acidic or basic functionalgroup and are, thus, capable of forming pharmaceutically-acceptablesalts with pharmaceutically-acceptable acids and bases. The term"pharmaceutically-acceptable salts" in these instances refers to therelatively non-toxic, inorganic and organic acid and base addition saltsof compounds of formula (1). These salts can be prepared by reacting thepurified compound with a suitable acid or base. Suitable bases includethe hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptablemetal cation, ammonia, or a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.Representative acid addition salts include the hydrobromide,hydrochloride, sulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthalate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.

The compounds of formula (1), and the pharmaceutically-acceptable saltsthereof, are inhibitors of β-lactamases. As discussed in the Background,it has previously been reported that boric acid and certain boronicacids are inhibitors of certain β-lactamases. These inhibitors aredifferent than the inhibitors of the invention defined by formula (1).Moreover, many of the compounds of formula (1) are much more effectiveinhibitors of β-lactamases than the prior art inhibitors (see theExamples below).

Assays for the inhibition of β-lactamase activity are well known in theart. For instance, the ability of a compound to inhibit β-lactamaseactivity in a standard enzyme inhibition assay may be used (see, e.g.,Example 2 below and M. G. Page, Biochem J. 295 (Pt. 1) 295-304 (1993)).β-lactamases for use in such assays may be purified from bacterialsources or, preferably, are produced by recombinant DNA techniques,since genes and cDNA clones coding for many β-lactamases are known. See,e.g., S. J. Cartwright and S. G. Waley, Biochem J. 221, 505-512 (1984).Alternatively, the sensitivity of bacteria known, or engineered, toproduce a β-lactamase to an inhibitor may be determined (see Example 3below). Other bacterial inhibition assays include agar disk diffusionand agar dilution. See, e.g., W. H. Traub & B. Leonhard, Chemotherapy43, 159-167 (1997). Inhibition includes both reduction and eliminationof β-lactamase activity.

The compounds of formula (1) are also effective against bacteriaresistant to β-lactam antibiotics as a result of porin mutations (see,e.g., Example 5 below). Porin mutations are mutations in the proteinswhich form porin channels in bacterial cell walls. These mutationsreduce the ability of β-lactam antibiotics to enter bacterial cells inwhich the mutations occur, thereby making the bacteria resitant to theseantibiotics.

The compounds of formula (1), or pharmaceutically-acceptable saltsthereof, can be used to treat β-lactam-antibiotic-resistant bacterialinfections. "β-lactam-antibiotic-resistant bacterial infection" is usedherein to refer to an infection caused by bacteria resistant totreatment with β-lactam antibiotics due primarily to the action of aβ-lactamase, a porin mutation, or both. Resistance to β-lactamantibiotics can be determined by standard antibiotic sensitivitytesting. The presence of β-lactamase activity can be determined as iswell known in the art (see above). The presence of a porin mutation canbe detected by polymerase chain reaction analysis of porin genes,polyacrylamide gel electrophoresis of a preparation obtained by mildosmotic shock (e.g., treatment with hypotonic solution containing EDTA,followed by gentle centrifugation and separation of the supernatant) ofthe bacteria (absence of a protein of the appropriate molecular weightbeing indicative of a porin mutation), or by determining resistance toinfection by bacteriophage Tu1A (a standard test for OmpF⁻ porinmutations). Alternatively, and preferably, the sensitivity of aparticular bacterium to the combination of a compound of formula (1), ora pharmaceutically-acceptable salt thereof, and a β-lactam antibioticcan be determined by standard antibiotic sensitivity testing methods.

To treat a β-lactam resistant bacterial infection, an animal sufferingfrom such an infection is given an effective amount of a compound offormula (1), or a pharmaceutically-acceptable salt thereof, and aneffective amount of a β-lactam antibiotic. The compound of formula (1),or a pharmaceutically-acceptable salt thereof, and the antibiotic may begiven separately or together. When administered together, they may becontained in separate pharmaceutical compositions or may be in the samepharmaceutical composition.

Many suitable β-lactam antibiotics are known. These includecephalosporins (e.g., cephalothin), penicillins (e.g., amoxicillin),monobactams (e.g., aztreonam), carbapenems (e.g., imipenem),carbacephems (loracarbef), and others. β-lactam antibiotics areeffective (in the absence of resistance) against a wide range ofbacterial infections. These include those caused by both gram-positiveand gram-negative bacteria, for example, bacteria of the genusStaphylococcus (such as Staphylococcus aureus and Staphylococcusepidermis), Streptococcus (such as Streptococcus agalactine,Streptococcus penumoniae and Streptococcus faecalis), Micrococcus (suchas Micrococcus luteus), Bacillus (such as Bacillus subtilis), Listerella(such as Listerella monocytogenes), Escherichia (such as Escherichiacoli), Klebsiella (such as Klebsiella pneumoniae), Proteus (such asProteus mirabilis and Proteus vulgaris), Salmonella (such as Salmonellatyphosa), Shigella (such as Shigella sonnei), Enterobacter (such asEnterobacter aerogenes and Enterobacter facium), Serratia (such asSerratia marcescens), Pseudomonas (such as Pseudomonas aeruginosa),Acinetobacter such as Acinetobacter anitratus), Nocardia (such asNocardia autotrophica), and Mycobacterium (such as Mycobacteriumfortuitum). Effective doses and modes of administration of β-lactamantibiotics are known in the art or may be determined empirically asdescribed below for the compounds of formula (1).

It has also been found that the compounds of formula (1), orpharmaceutically-acceptable salts thereof, are antibacterial bythemselves, although at higher concentrations than β-lactam antibiotics.Indeed, they have shown activity against β-lactam-antibiotic-resistantbacteria. Although not wishing to be bound by any particular theory, itis believed that this antibacterial activity is due to the binding ofthe inhibitors to PBPs which resemble β-lactamases. Since PBPs are foundin all bacterial species susceptible to β-lactam antibiotics, it isexpected that the compounds of formula (1), orpharmaceutically-acceptable salts thereof, will be effective against thesame bacteria as the β-lactam antibiotics (see above). As with theβ-lactam antibiotics, sensitivity of bacteria to the compounds offormula (1), or pharmaceutically-acceptable salts thereof, can bedetermined by standard antibiotic sensitivity testing.

To treat an animal suffering from a bacterial infection, includingβ-lactam-antibiotic-resistant bacterial infections, an effective amountof a compound of formula (1), or a pharmaceutically-acceptable saltthereof, is administered to the animal, alone or in combination with aβ-lactam antibiotic. Effective dosage forms, modes of administration anddosage amounts of a compound of formula (1), may be determinedempirically, and making such determinations is within the skill of theart. It is understood by those skilled in the art that the dosage amountwill vary with the activity of the particular compound employed, theseverity of the bacterial infection, whether the bacterial infection isresistant to treatment with β-lactam antibiotics, the route ofadministration, the rate of excretion of the compound, the duration ofthe treatment, the identity of any other drugs being administered to theanimal, the age, size and species of the animal, and like factors wellknown in the medical and veterinary arts. In general, a suitable dailydose will be that amount which is the lowest dose effective to produce atherapeutic effect. The total daily dosage will be determined by anattending physician or veterinarian within the scope of sound medicaljudgment. If desired, the effective daily dose of a compound of formula(1), or a pharmaceutically-acceptable salt thereof, may be administeredas two, three, four, five, six or more sub-doses, administeredseparately at appropriate intervals throughout the day. Treatment of abacterial infection, including β-lactam-antibiotic-resistant bacterialinfections, according to the invention, includes mitigation, as well aselimination, of the infection.

Animals treatable according to the invention include mammals. Mammalstreatable according to the invention include dogs, cats, other domesticanimals, and humans.

Compounds of formula (1) or pharmaceutically-acceptable salts thereof,may be administered to an animal patient for therapy by any suitableroute of administration, including orally, nasally, rectally,intravaginally, parenterally, intracisternally and topically, as bypowders, ointments or drops, including buccally and sublingually. Thepreferred routes of administration are orally and parenterally.

While it is possible for the active ingredient(s) (one or more compoundsof formula (1), or pharmaceutically-acceptable salts thereof, alone orin combination with a β-lactam antibiotic) to be administered alone, itis preferable to administer the active ingredient(s) as a pharmaceuticalformulation (composition). The pharmaceutical compositions of theinvention comprise the active ingredient(s) in admixture with one ormore pharmaceutically-acceptable carriers and, optionally, with one ormore other compounds, drugs or other materials. Each carrier must be"acceptable" in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient.

Pharmaceutical formulations of the present invention include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal and/or parenteral administration. Regardless of theroute of administration selected, the active ingredient(s) areformulated into pharmaceutically-acceptable dosage forms by conventionalmethods known to those of skill in the art.

The amount of the active ingredient(s) which will be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration andall of the other factors described above. The amount of the activeingredient(s) which will be combined with a carrier material to producea single dosage form will generally be that amount of the activeingredient(s) which is the lowest dose effective to produce atherapeutic effect.

Methods of preparing pharmaceutical formulations or compositions includethe step of bringing the active ingredient(s) into association with thecarrier and, optionally, one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing theactive ingredient(s) into association with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of the activeingredient(s). The active ingredient(s) may also be administered as abolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient(s) is/are mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered activeingredient(s) moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient(s) thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredients) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient(s) can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the active ingredients)include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient(s), the liquid dosage forms may contain inert diluentscommonly used in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active ingredient(s), may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing the active ingredient(s) with one ormore suitable nonirritating excipients or carriers comprising, forexample, cocoa butter, polyethylene glycol, a suppository wax orsalicylate and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the rectum or vaginal cavityand release the active ingredient(s). Formulations of the presentinvention which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of the activeingredient(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The activeingredient(s) may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to theactive ingredient(s), excipients, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active ingredient(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of the active ingredient(s) to the body. Such dosage forms canbe made by dissolving, dispersing or otherwise incorporating the activeingredient(s) in a proper medium, such as an elastomeric matrixmaterial. Absorption enhancers can also be used to increase the flux ofthe active ingredient(s) across the skin. The rate of such flux can becontrolled by either providing a rate-controlling membrane or dispersingthe active ingredient(s) in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise the active ingredient(s) in combination with oneor more pharmaceutically-acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,solutes which render the formulation isotonic with the blood of theintended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the activeingredient(s), it is desirable to slow the absorption of the drug fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility. The rate of absorption of the activeingredient(s) then depends upon its/their rate of dissolution which, inturn, may depend upon crystal size and crystalline form. Alternatively,delayed absorption of parenterally-administered active ingredient(s) isaccomplished by dissolving or suspending the active ingredients) in anoil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe active ingredient(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of the activeingredient(s) to polymer, and the nature of the particular polymeremployed, the rate of release of the active ingredient(s) can becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsare also prepared by entrapping the active ingredient(s) in liposomes ormicroemulsions which are compatible with body tissue. The injectablematerials can be sterilized for example, by filtration through abacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The pharmaceutical compositions of the present invention may also beused in the form of veterinary formulations, including those adapted forthe following: (1) oral administration, for example, drenches (aqueousor non-aqueous solutions or suspensions), tablets, boluses, powders,granules or pellets for admixture with feed stuffs, pastes forapplication to the tongue; (2) parenteral administration, for example,by subcutaneous, intramuscular or intravenous injection as, for example,a sterile solution or suspension or, when appropriate, by intramammaryinjection where a suspension or solution is introduced into the udder ofthe animal via its teat; (3) topical application, for example, as acream, ointment or spray applied to the skin; or (4) intravaginally, forexample, as a pessary, cream or foam.

EXAMPLES Example 1

Identification of Potential β-Lactamase Inhibitors

To target sites on the E. coli TEM-1 and AmpC β-lactamases to whichnovel inhibitors might bind, the structures of certain enzyme-inhibitorcomplexes were determined. These structures and other known structuresof enzyme-inhibitor and enzyme-substrate complexes were used to definethe binding sites of the enzymes. Additional potential binding sites onAmpC were identified using computational methods.

TEM-1 and AmpC were chosen because, as discussed above, these twoβ-lactamases are responsible for resistance to β-lactam antibiotics inE. coli, and the versions of TEM and AmpC in other bacterial speciesshare high sequence identity and are structurally similar to TEM-1 andAmpC from E. coli. The high degree of species similarity among the TEMand AmpC β-lactamases suggests that inhibitors discovered for the E.coli enzymes will be active against Types I and II 62 -lactamases inother bacterial species. This is consistent with the antimicrobial datapresented below which show that the boronic acid derivatives of theinvention are active against bacteria expressing several different typeI and type II TEM-1 β-lactamases (e.g., the AmpC-like enzyme expressedby Enterobacter cloacae).

AmpC was expressed in E. coli JM109 cells in which the native AmpC genewas attenuated or completely removed (obtained from Larry Blaszczak, EliLilly and Co., Indianapolis, Ind.). DNA coding for the enzyme waslocated on a plasmid under the control of a temperature sensitiverepressor. Cells containing this plasmid were grown in 2 liters of LBbroth in a fermentor to log phase. Enzyme expression was then induced bytemperature shock, and the cells were allowed to grow overnight. AmpCprotein was purified from the supernatant over an Affigel-10 aminophenylboronate affinity column (Bio-Rad Laboratories, 1000 Alfred Nobel Drive,Hercules, Calif.). The purity of the sample was estimated by HPLC to be96% or better. The amount of enzyme produced was estimated to be 150 mgbased on absorbance at 280 nm.

Protein from this preparation was used to grow diffraction-qualitycrystals. The structures of three boronate-enzyme complexes(m-aminophenylboronic acid (MABP), benzo[b]thiophene-2-boronic acid(BZBTH2B) and m-nitrophenylboronic acid (3NPB)) were also determined.Protein crystals were obtained by vapor diffusion using a hanging dropmethod. The concentration of protein in the drops was 3-6 mg/ml, and theconcentration of the boronic acid inhibitor was 1-10 mM. The buffer inthe well was 1.7 M potassium phosphate, pH 8.7. For the MAPB-AmpCcomplex, 2% methane pentane diol was also used. For the MAPB-AmpCcomplex, x-ray diffraction data were collected on a Xuong-Hamlinmultiwire detector. For the BZBTH2B-AmpC and 3NPB-AmpC complexes, datawere collected on an R-axis image plate system. The structure of theMAPB-AmpC complex was refined with the program TNT (D. E. Tronrud, ActaCrystallogr. Sect. A. 48 912-916 (1992)). The structures of theBZBTH2B-AmpC and 3NPB-AmpC complexes were refined with the programX-Plor (Brunger, A. T., X-PLOR Version 3.1 A System For X-rayCrystallography And NMR (Yale University Press, New Haven, Conn., 1992).For all three boronic acid-AmpC complex structures, models were builtwith the program 0 (Jones et al., Acta Crystallogr. Sect A 47, 110-119(1991)). The x-ray crystallographic statistics for these three complexesare given in Table 1 below. The structures of the three boronatecomplexes and the structure of the phosphonate complex of Knox andcolleagues (Lobkovsky, et al., Biochemistry, 33, 6762-6772 (1994)) wereused to partly define the binding sites of AmpC.

For TEM-1 the structures of three inhibitor-enzyme complexes determinedby Natalie Strynadka (Strynadka et al., Nature, 359, 700-705 (1992);Strynadka et al., Nature Structural Biology, 3, 233-239 (1996);Strynadka et al., Nat. Struct. Biol., 3, 688-695 (1996)) were used todefine the binding sites of TEM-1. These complexes were aprotein-protein complex involving a β-lactamase inhibitory protein, acomplex with penicillin G, and a complex with a boronate inhibitor.

Using the computational methods of Kuntz (Kuntz et al., J. Mol. Biol.,161, 269-288 (1982)) and Honig (Gilson and Honig, Nature, 330, 84-86(1987)), additional potential binding sites in a tunnel region of AmpCthat the various inhibitors did not take advantage of, but which seemedto be present in the structure of the enzyme, were identified.

Using the binding sites defined as described above, other boronic acidswere identified as potential inhibitors of β-lactamase. 2-Phenylboronicacid, MABP, thiophene-2-boronic acid (TH2B), 3NPB, and4,4'-biphenyldiboronic acid (BIPD) were selected as a representativesample of commercially-available boronic acids for modeling into theAmpC active site. Structures and conformational libraries for eachcompound were created using the Sybyl molecular modeling suite (TriposInc., St. Louis, Mo.). Conformer interactions with AmpC were scoredbased on steric and electrostatic criteria using the DISTMAP (Shoichet,B. K.; Bodian, D. L.; Kuntz, I. D., J. Comp. Chem., 13, 380-397 (1992))and DelPhi (Gilson, M. K.; Honig, B. H., Nature, 330, 84-86 (1987))programs (Table 1). Two major families of ligand orientations wereidentified. In one "MAPB-like" mode, the boronic acid ligand is orientedsimilarly to the inhibitor in the MAPB-AmpC structure and is predictedto interact with residues Thr316, Asn346, and Asn289. In a second"phosphonate-like" mode, the boronic acid ligand is oriented similarlyto the phosphonate ligand in an AmpC-inhibitor complex determined byLobkovsky, et al. (Lobkovsky, E.; Billings, E. M.; Moews, P. C.; Rahil,J.; Pratt, R. F.; Knox, J. R. Biochemistry, 33, 6762-6772 (1994)) and ispredicted to interact with residues Asn152 and Gln120. The numberingscheme used to refer to E. coli AmpC residues is that of Galleni et al.Galleni et al., Sequence and Comparative Analysis of Three Enterobactercloacae ampC β-Lactamase Genes and Their Products, Biochem. J.250:753-760 (1988).

Several predictions arose out of these modeling studies. Thedistribution of orientations in the two modes was generally correlatedwith the size of the boronic acid ligand, with larger ligands favoringthe "phosphonate-like" mode because of steric clashes with the receptorin the "MAPB-like" mode. Ligands such as TH2B, which might bind in an"MAPB-like" conformation, would be expected to have specificinteractions with features of the AmpC binding site that might improvetheir potency relative to AmpC. Larger ligands, such as BZBTH2B, wereexpected to bind in the "phosphonate-like" geometry. In this lattergeometry, ligands such as BZBTH2B and 3NPB would be expected to interactwith Gln120, Asn152 and Tyr150.

The geometries of BZBTH2B and 3NPB in complex with AmpC, determined byx-ray crystallography, are consistent with these predictions. A refinedstructure of BZBTH2B (2Fo-Fc electron density) showed the inhibitorcovalently connected to serine 64 (Ser64). The density clearly definedthe orientation of this compound in the binding site. Thecrystallographic statistics were good (R-factor 0.179, Rfree 0.229), andall bond and angle values fell within the allowed deviations for awell-refined structure. A similar refined structure of 3NPB (2Fo-Fcelectron density) showed that this inhibitor was also covalentlyconnected to Ser64. FIG. 5 shows key active site residues of the enzymeenvironment around BZBTH2B. Residues displayed were typically within 5angstroms of BZBTH2B, with the exception of Arg349 and Asn346. Theselast two residues are nevertheless part of a polar network thatinteracts with the O2 hydroxyl of BZBTH2B through two well-defined watermolecules (small spheres). The dotted lines indicate hydrogen bondinteractions, with atoms within 2.6-3.2 angstroms of each other. Theatomic resolution nature of these x-ray structures clearly showed theinteractions that these inhibitors are making with AmpC and provide astrong framework for identifying and designing future boronic acidinhibitors of the enzyme.

                                      TABLE 1                                     __________________________________________________________________________                           R-factor                                                                           Space Group,                                      Inhibitor                                                                           Resolution                                                                         Data        R-free                                                                             cell dimensions                                   complex                                                                             range                                                                              Completeness                                                                         R-merge                                                                            (%)  (Å; deg.)                                     __________________________________________________________________________    BZBTH2B                                                                             20-2.25                                                                            87%    9.4  17.9, 22.4                                                                         C2; a = 119, b = 78, c = 99                                                   α = γ = 90; β = 116              3NPB  20-2.15                                                                            95%    8.4  22, 25                                                                             C2; a = 121, b - 78, c = 100                                                  α = γ = 90; β = 117              MAPB  20-2.3                                                                             95%    8.8  19.5,                                                                              C2; a = 119, b = 77, c = 98                                              unknown                                                                            α = γ = 90.0; β = 116            __________________________________________________________________________

Structural modeling was followed by testing for inhibition of enzymeactivity and antibacterial activity. This was followed by modeling andtesting of additional compounds. For testing results, see Examples 2-5.This cycle of structural modeling, enzymatic testing and antibacterialevaluation led to the following observations.

An intriguing feature of the MAPB--AmpC complex is how few obviouslyfavorable interactions are observed between the aryl group of theinhibitor and the enzyme. Still, MAPB has a K_(i) of 7.3±0.9 μM forAmpC. One possible explanation for the affinity of MAPB is that thebinding of the compound is driven by ligand hydrophobicity. To testthis, the inhibition of several other hydrophobic boronic acids wasmeasured. Both 1-naphthyl- and 9-phenanthrene- boronic acids have two tothree-fold worse (higher) dissociation constraints than MAPB. Theseligands are larger than MAPB, so the effect of hydrophobicity may becomplicated by steric constraints. 2-naphthylboronic acid has anaffinity (K_(i) =8.5±1.8 μM) comparable to MAPB, suggesting that thepresence of a larger hydrophobic substituent in the right orientationdoes not hinder binding. Certainly for diphenylboronic acid, which lacksmeasurable inhibition of AmpC, modeling suggests potential stericconflicts with residues Tyr150 and Lys67. On the other hand, the smallerand more flexible n-butylboronic acid should have little difficultyfitting into the AmpC site, and yet it also displays no measurableinhibition of AmpC. Taken together, these results suggest that boronicacids must have the correct stereochemical arrangement of functionality;hydrophobicity alone is not sufficient to explain affinity.

The differential affinities of the boronic acids for AmpC might be dueto activation of the boronic acid group as an electrophile bysubstituents on the aryl ring. The affinities of 2-formyl- and4-formyl-phenylboronic acids, and those of 3-trifluoro- and4-trifluoro-phenylboronic acids were compared. If affinity was modulatedmostly by effects on electrophilicity of the boronic acid group, onemight expect electron withdrawing groups at the 2 and 4 positions to beabout equal activators, but both to be better than similar groups at the3 position. Instead, it was found that derivatives at the 3 positionwere more active than ones at 4, and that groups at 2 are much lessactive than either derivatives at 3 or 4. This is consistent with thesteric constraints around the 2 position of MAPB and the polarenvironment around position 3 in the "MAPB-like" orientation. It wasalso found that the (S) and (R) stereoisomers of 3-tetrahydrofuranylboronic acid differ in affinity for AmpC by an order of magnitude. Thiscan only be explained by differential non-covalent interactions with theenzyme, perhaps with the nearby Thr316 residue if these ligands assume a"MAPB-like" binding mode.

Perturbations that maintained the overall steric disposition of MAPB,but changed its functionality, were next considered. In the case of a"MAPB-like" binding mode, hydrogen-bonding groups from Asn289, Asn346,and Arg349 are in proximity to potential ring substituents at the 3- and4-positions of a MAPB-like compound. Alternatively, if the boronic acidligand binds in a "phosphonate-like" orientation, hydrogen-bondinggroups from Gln120 and Asn152 lie nearby. It should be noted that, withthe many approximations used in the boronic acid-based ligand modeling(no allowance for enzyme flexibility, simplistic modeling of ligandelectrostatic properties, etc.), these results should only be used as aguide. In the case of either binding mode, it seemed reasonable to lookfor ligand functionality that could be involved in hydrogen bonds with,or at least offer polar complements to, these nearby residues.Consistent with these structural considerations, the K_(i) values of the3-nitro-, and 3-trifluoro derivatives of phenylboronic acid are in theone to two micromolar range, three- to five-fold better than MAPB.However, the 3-carboxy derivative of phenylboronic acid does not displaysignificant affinity for AmpC (K_(i) >100 μM). This suggests that thiscompound cannot bind to AmpC in a manner that allows interaction betweenthe m-substituent of the inhibitor and the Arg349 residue of the enzyme.

Three regions of the enzyme were of particular interest. Assuming a"MAPB-like" binding mode for boronic acid-based ligands, a "canyon" wasnoted near position 3 of the MAPB ring in the crystallographic complex.In addition, a large hydrophilic tunnel, about 15 Å in length, was notednear the 4-position of MAPB in the crystallographic complex that ranthrough the surface of the enzyme. Alternatively, if, as the modelingresults suggest, a potential boronic acid ligand may also adopt a"phosphonate-like" binding orientation, the pocket defined by residuesAla318, Tyr221, Gln120, and Asn152 offers the potential for increasingligand interactions with the target site by changing ligandfunctionality.

Surprisingly, the 4,4'-biphenyldiboronic acid analog inhibits AmpCpotently, with a K_(i) of 0.18±0.02 μM. Although this derivative may bemodeled to fit near the mouth of the tunnel region of AmpC notedearlier, it cannot do so without accommodation on the part of theenzyme. If the same mode of binding as MAPB is assumed, without enzymerelaxation, this inhibitor would come into close contact with Ser287,Asp288, Asn289, and Asn346. Although modeling results suggest otherconformations are possible, none of them make interactions that clearlyexplain the affinity of this compound. The most conservative explanationis that the 4,4'-biphenyldiboronic acid analog maintains the overalldisposition of groups suggested by the MAPB-E. coli complex. This wouldlead to interactions with the mouth of the tunnel region, includingresidues such as Ala292. However, in order to do so, residues Asn346 andSer287 would have to move slightly away from the ligand.

The proximity of the hydroxyl groups of Tyr150 and Thr316 to ring atomsof MAPB in the crystallogrphic complex suggested that polar orpolarizable atoms at positions 2 or 3 might better complement the enzymethan the phenyl ring of MAPB. Consistent with this view,thiophene-2-boronic acid was found to have a K_(i) of 2.5±0.4 μM againstAmpC, and (R)-3-tetrahydrofuranyl-boronic acid was found to have a K_(i)of 1.4±0.1 μM. The thiopene-3-boronic acid, which should be unable toaccept a hydrogen bond from Tyr150 in a "MAPB-like" binding orientation,has a much worse affinity for AmpC (K_(i) =22.1±3.5 μM). The(S)-3-tetrahydrofuranylboronic acid, the heteroatom of which, in a"MAPB-like" binding orientation should be unable to interact withThr316, has a K_(i) of 15.8±0.8 μM. On the other hand, the poor affinityof 2-furanylboronic acid (K_(i) >>100 μM), which, like the 2-thiophenederivative, should be able to accept a hydrogen bond from Tyr150, isdifficult to explain simply based on hydrogen bonding considerations.The differential activity of the 2-thiophene derivative relative to the2-furanyl derivative might reflect subtle differences in polarity andpolarizability of an aryl sulfur versus an aryl oxygen. Alternatively,the difference in activities might reflect the different shapes of themolecules.

It was also considered that substitutions, including the presence oflarger heteroaryl groups, might improve the potency of TH2B. Modelingsuggested that although ligands containing larger systems likebenzo[b]heteroarylboronic acids probably would not fit into the AmpCsite in a "MAPB-like" binding mode (based on orientation distributionsand assuming no accommodation on the part of the enzyme), thesecompounds should still be able to bind to the enzyme in other productiveorientations. Several derivatives of TH2B were tested, the most potentof which was benzo[b]thiophene-2-boronic acid. This compound has a K_(i)of 27 nM for AmpC.

Benzo[b]thiophene-2-boronic acid is approximately 200-fold more activethan thiophene-2-boronic acid, suggesting that interactions with thesecond aryl ring contribute considerably to affinity. This inference issupported by the activity of benzo[b]furan-2-boronic acid, which isabout 1000-fold more active than the furan-2-boronic acid parent. At thesame time, a comparison of the activity of BZBTH2B with2-naphthylboronic acid (K_(i) =8.5±1.8 μM), which should place itsdistal aryl ring in approximately the same area as the benzo[b]thiophenederivative, confirms the importance of the thiophene ring. Modelbuilding suggests that BZBTH2B may bind to AmpC in the pocket defined byresidues Gln120, Asn152, and Tyr218.

The wide variety of chemical functionality present in the boronic acidcompounds tested has allowed mapping of the AmpC binding site andsuggested modifications to improve the potency of the agents tested.Modeling of these inhibitors suggests that they may be interacting withthe enzyme in ways unanticipated by earlier classes of inhibitors. Atthe same time, it must be admitted that such modeling carries with itsome ambiguity and key questions regarding the structural bases foractivity remain unanswered.

Example 2

Testing of Compounds for Inhibition of β-Lactamases

Compounds were tested for inhibition of TEM-1 and AmpC β-lactamases fromE. coli using a spectrophotometric assay (Page, Biochem. J., 295,295-304 (1993)). AmpC was prepared as described in Example 1. TEM-1 wasprovided by Natalie Strynadka, University Of Alberta, Edmonton, Canada.Alternatively, TEM-1 may be produced as follows. The TEM-1 gene iscloned into HpaI site of pALTER-EX2 (Promega). The gene is under controlof the T7 promoter which is turned on for protein expression. TEM-1 maybe expressed in JM109 cells, as well as several other E. coli strains.Cells are grown to late log phase, followed by induction of proteinexpression. The cells are spun down and the supernatant, into which theenzyme has been exported, is collected. Because the enzyme has beenexported into the supernatant, purification may be achieved usingstandard column chromatography, as described in Matagne et al. BiochemJ. 265, 131-146 (1990); Escobar et al, Biochemistry 33, 7619-7626(1994).

Initial stock solutions of 1-100 mM concentrations of each compound tobe tested were prepared in DMSO (dimethyl sulfoxide). Solubility andabsorbance profiles were determined by incremental addition of smallvolumes of DMSO stock solutions to assay buffer (50 mM phosphate, pH7.0) at 25° C. using an HP8543 UV/Visible spectrophotometer withmulti-cell transport running HP ChemStation software (version 2.5).Enzymatic testing was typically started at an upper concentration limitdetermined by the solubility and absorbance profile of the compound.

Standard assay conditions for AmpC were as follows: pH 7.0; 100 μMcephalothin, sodium salt, as substrate; reaction monitored at 265 nm(cephalothin β-lactam absorbance peak); T=25° C.; 50 mM phosphatebuffer; no incubation of inhibitor with enzyme; cycle times of 10-15seconds; total reaction volume=1 mL; run time=5 minutes; reactioninitialized with addition of 0.06 nM AmpC. The background rate ofcephalothin hydrolysis under these conditions was found to be two tothree orders of magnitude less than the rate of the enzyme-mediatedcephalothin hydrolysis, so no correction for background hydrolysis ofsubstrate was used. For TEM-1, 100 μM 6-β-furylacryloylamidopenicillanicacid, triethylammonium salt (FAP), was used as the substrate, thereaction was monitored at 340 nm (FAP β-lactam absorbance peak) and thecycle time was increased to 25 seconds (since this substrate wassomewhat light sensitive). Due to the light sensitivity of FAP, thebackground rate of hydrolysis for this substrate was found to beminimal, but not insignificant, so all measured control and inhibitedcell rates were corrected by subtraction of the FAP background rate. Allother conditions for the TEM-1 assays were identical to those for theAmpC assays. DMSO was added to enzyme controls in all cases. Standard 1mm path length quartz spectrophotometric cells (Hellma Cells, Inc.,Jamaica, N.Y.) were used in the assays. All assays were performed on thesame HP8543 spectrophotometer noted earlier.

Linear and quadratic fits to the absorbance data for the full timecourse of each reaction were used to determine the reaction rate foreach spectrophotometric cell. The resulting reaction rate data were usedto calculate the inhibition constants for each potential inhibitor usingthe method of Waley (S. G. Waley, Biochem. J. 205, 631-633 (1982)).Briefly, this method involves the use of the integrated Michaelis-Mentenequation to calculate K_(i) values for enzyme inhibitors from acomparison of the reaction rates of uninhibited and inhibited enzymaticreactions.

Specificity testing was performed by assaying the activity of aninhibitor against α-chymotrypsin (bovine pancreatic), β-trypsin (bovinepancreatic), and elastase (porcine pancreatic). Substrates forα-chymotrypsin (N-benzoyl-L-tyrosine ethyl ester, BTEE) and β-trypsin(N-benzoyl-L-arginine ethyl ester, BAEE) were purchased from SigmaChemical, St. Louis, Mo. The elastase substrate used (elastase substrate1, Nα-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, was purchased fromCalbiochem, San Diego, Calif. All enzymes used for specificity testingwere purchased from Sigma Chemical, St. Louis, Mo. For α-chymotrypsin, 3μl of a 1 mg/ml enzyme stock solution (50 mM phosphate buffer, pH 7) wasincubated with the boronate being tested for 5 minutes; then thereaction was initialized by addition of 630 μM BTEE from a DMSO stocksolution. The reaction was performed at 25° C. and monitored at 260 nm.For β-trypsin, 40 μl of a 0.8 mg/ml enzyme stock solution (50 mMphosphate buffer, pH 7) was incubated with the boronate being tested for5 minutes; then the reaction was initialized by addition of 600 μM BAEEfrom a DMSO stock solution. For elastase, 50 μl of a 1 mg/ml enzymestock solution (50 mM phosphate buffer, pH 7) was incubated with theboronate being tested for 5 minutes; then the reaction was initializedby addition of 64 μM elastase substrate 1 from a DMSO stock solution.

The compounds tested are those listed in Tables 2A and 2B below. Certainprior art compounds (marked with an * in Table 2A) were tested forcomparative purposes. 9-Phenanthreneboronic acid (9PHNB) was obtainedfrom TCI America, Portland, Oreg. Butylboronic acid (BUTB),4-bromophenylboronic acid (4BPB), 3-nitrophenylboronic acid (3NPB),2-hydroxy-5-(3-(trifluoromethyl)phenylazo) benzeneboronic acid (HFAB),2,4,6-tris(5-(4-bromophenylazo)-2-hydroxyphenyl) boroxin (4BPAPB), anddiethanolamine-(3R)-(+)-tetrahydrofurnylboronate (DETHFB) were obtainedfrom Aldrich Chemical, Milwaukee, Wis. (HFAB and 4BPAPB are products ofthe Sigma-Aldrich Library of Rare Chemicals). The remaining compoundstested were obtained from Lancaster Synthesis, Windham, N.H. Allcompounds were used as is with no additional purification orverification performed.

The results of the testing are presented in Tables 2A, 2B and 2C below.Tables 2A and 2B contain the results of the assays of inhibition of AmpCand TEM-β-lactamases, and Table 2C contains the results of thespecificity testing. In the tables, N.T.=not tested, and N.A.=not activeat the maximum inhibitor concentration tested. Other abbreviations usedin Tables 2A, 2B and 2C which are not explained in this example areexplained in FIGS. 1A, 1D and 1E.

                  TABLE 2A                                                        ______________________________________                                                       Ki E. coli AmpC                                                                           Ki E. coli TEM-1                                   boronate       (μM)     (μM)                                            ______________________________________                                        borinic acids                                                                 DFB            >500        >>100                                              acyclic alkylboronates                                                        BUTB           >500        >100                                               heterocyclic alkylboronates                                                   RDETHFB        1.1         27.0                                               SDETHFB        15.0        86.2                                               arylboronates                                                                 BIPD           0.6         >>100                                              HFAB           1.3         N.T.                                               3TFMB          1.6         85.0                                               NSULFB*        1.6         88.0                                               3NPB           1.9         24.0                                               4BPB           2.6         31.3                                               4FORMB*        2.8         35.0                                               4MEPB*         5.2         >100                                               MAPB*          5.8         >>100                                              4COOHB         5.8         >>100                                              4FB            6.1         >>100                                              B14DA          6.9         40.0                                               4BPAPB         7.2         1.2                                                4MEOB          7.7         >100                                               2FDB*          8.0         >100                                               4TFMB          9.0         6.3                                                NAPB           10.4        34.0                                               9PHNB          12.6        31.0                                               2FORMB*        62.0        >100                                               heterocyclic arylboronates                                                    TH2B           3.3         31.0                                               TH3B           17.0        100.0                                              ______________________________________                                    

                  TABLE 2B                                                        ______________________________________                                                     Ki E. coli AmpC                                                                           Ki E. coli TEM-1                                     boronate     (μM)     (μM)                                              ______________________________________                                        BZBTH2B       0.04       4.0                                                  BZBF2B        0.07       8.0                                                  5CLTH2B      1.4         17.0                                                 5ACTH2B      1.8         >50                                                  TH2B         3.3         31.0                                                 3FTH2B       3.5         N.I.                                                 ______________________________________                                         N.I. = no inhibition observed at an inhibitor concentration of 100 μM.

                  TABLE 2C                                                        ______________________________________                                                IC50 (μM) for:                                                     boronate  AmpC    CHT         TRY   ELST                                      ______________________________________                                        TH2B      10.0    >200.0      >200.0                                                                              100.0                                     ______________________________________                                         CHT = alphachymotrypsin, bovine pancreas;                                     TRY = betatrypsin, bovine pancreas;                                           ELST = elastase, porcine pancreas                                        

Example 3

Antibacterial Activity

Bacterial cell culture testing was performed and interpreted followingthe guidelines of the National Committee for Clinical LaboratoryStandards (National Committee for Clinical Laboratory Standards. Methodsfor Dilution Antimicrobial Susceptibility Tests for Bacteria that GrowAerobically. Approved Standard M7-A3. National Committee for ClinicalLaboratory Standards, Villanova, Pa. 1993). After incubation, the growthof the cells was visually inspected. The minimum inhibitoryconcentration (MIC) is the lowest concentration where there no cellgrowth was observed. The results are presented in the tables below.

The following strains were used: Enterobacter cloacae cell line withderepressed β-lactamase production (Ent-Der), and Escherichia coliRYC1000 (araD139 D lacU169 rpsL D rib7 thiA gyrA recA56) cell lines,harboring the plasmid pBGS19 (with no β-lactamase), or the β-lactamasecontaining plasmids pBGAmpC (AmpC β-lactamase from E. coli; Eco-AmpC),or pBGAmpC-MHN (AmpC β-lactamase from Enterobacter cloacae;Eco-AmpCEnt). Plasmids pBGAmp-MHN and pBGAmpC were constructed by PCRamplification of the respective E. cloacae and E. coli chromosomal ampCgenes and subsequent cloning into pBGS18 (Spratt, B. G.; Hedge, P. I.;Heesen, S.; Edelman, A.; Broome-Smith, J. K. Gene 41, 337-342 (1986)).TEM-10 and TEM-24 are mutants of TEM-1. TEM-10 and TEM-24 differ fromTEM-1 due to the following point substitutions: TEM-10 (R164S, E240K);TEM-24 (L102K, L162S, S235T, A237K). TEM-10 and TEM-24 also differ fromTEM-1 in being extended spectrum enzymes (i.e., they react with agreater range of substrates than TEM-1). Also tested were clinicalisolates of Pseudomonas aeruginosa. All bacterial strains and plasmidsare available from Jesus Blazquez and Fernando Baquero, Servicio deMicrobiologia, Hospital Ramon y Cajal, National Institute of Health,Madrid, Spain.

Boronic acid inhibitors were tested over a range of concentrations up toa maximum of 128 μg/ml. Several ratios, including 1:1, 2:1, 4:1, and1:3, of β-lactam antibiotic (amoxicillin (AX) or ceftazidime (CAZ)) toboronic acid compound were used in the assays. Tazobactam (TAZO), aclinically used β-lactamase inibitor, was used as a positive control.

                                      TABLE 3A                                    __________________________________________________________________________    Activity of boronic acid derivatives against bacterial cells in               combination with ceftazidime.                                                 MICs of ceftazidime (CAZ) and ceftazidime plus inhibitor (proportion:         4/1) in pg/ml. Strains used were: E. coli                                     RYC1000 (which does not produce β-lactamase) harboring plasmid           pBGS19 (which produces no β-lactamase), same                             strain harboring plasmid pBGTEM-24 (coding for TEM-24 β-lactamase, a     mutant of TEN-1) or plasmid PBGAmpC-                                          MHN (coding for AmpC β-lactamase of Enterobacter cloacae), and a         β-lactamase derepressed strain of Enterobacter                           cloacae (Ent. Der.). "Tazo" is tazobactam, a clinically used                  β-lactam-based β-lactamase inhibitor                                (Lederle Laboratories, Pearl River, NY).                                      CAZ      BIPD                                                                              9PHNB                                                                             DETHFB                                                                             3NBP                                                                              TH2B                                                                              48PAPB                                                                             BZBTH2B                                                                             5CLTH2B                                                                            TAZO                            __________________________________________________________________________    pBGS19                                                                             <0.5                                                                              <0.12                                                                             <0.25                                                                             0.5  <0.25                                                                             <0.25                                                                             <0.25                                                                              <0.25 <0.25                                                                              <0.25                           TEM-24                                                                             256 256/64                                                                            256/64                                                                            256/64                                                                             126/32                                                                            126/32                                                                            256/64                                                                             128/32                                                                              128/32                                                                             8/2                             AmpC-                                                                              32  8/2 8/2 4/1  4/1 8/2 8/2  2/0.5 4/1  4/1                             MHN                                                                           Ent. Der.                                                                          512 128/32                                                                            512/128                                                                           32/8 32/8                                                                              32/8                                                                              512/128                                                                            32/8  32/8 32/8                            __________________________________________________________________________

                                      TABLE 3B                                    __________________________________________________________________________    Activity of boronic acid derivatives against                                  bacterial cells when used by themselves.                                      MICs of inhibitors when used alone without ceftazidime,                       in pg/ml. The strains and plasmids are the same as for Table 3A.              BIPD     9PHNB                                                                             DETHFB                                                                             3NPB                                                                              TH2B                                                                              4BPAPB                                                                             BZBTH2B                                                                             5CLTH2B                                  __________________________________________________________________________    PBGS19                                                                             >256                                                                              128 >512 64  128 128  512   128                                      TEM-24                                                                             >256                                                                              128 >512 128 128 256  512   128                                      AmpC-                                                                              >256                                                                              256 >512 256 128 256  512   128                                      MHN                                                                           Ent. Der.                                                                          >256                                                                              >512                                                                              >512 >512                                                                              256 >512 512   128                                      __________________________________________________________________________

                                      TABLE 4A                                    __________________________________________________________________________    Activity of boronic acid derivatives against bacterial cells in               combination with amoxicillin.                                                 MICs of amoxicillin (AX) and amoxicillin plus inhibitor (proportion: 4/1)     in ug/ml.                                                                     Strains used were: E. coli RYC1000 harboring plasmid pBGS19 producing no      β-lactamase                                                              (EC), the same harboring pBGTEM-1 producing TEM-1 β-lactainase           (EC-T1), harboring                                                            pBGTEM-10 producing TEM-10 β-lactamase, a mutant of TEM-1 (EC-T10),      harboring                                                                     pBGAmpC-MHN (EC-AmpCEn), harboring pBGAmpC-E. coli producing AmpC (ECR-       AmpCEc), harboring a plasmid coding for a mutant of AmpC from                 Enterobacter                                                                  (ECR-AmpCEnM), and a β-lactamase derepressed strain of Enterobacter      cloacae (Ent-Der).                                                                    AX  BIPD                                                                              DETHFB                                                                             9PHNB                                                                              3NPB                                                                              TH2B TAZO                                       __________________________________________________________________________    EC                                                                            EC-T1   >2,048                                                                            256 512  512  256 128  8                                          EC-T10  >2,048                                                                            256 512  512  256 256  4                                          EC-AmpCEn                                                                             >2,048                                                                            128 32   256  64  32   16                                         ECR-AmpCEc                                                                            >2,048                                                                            128 128  512  64  64   32                                         ECR-AmpCEnM                                                                           >2,048                                                                            128 64   64   32  32   16                                         Ent-Der.                                                                              >2,048                                                                            256 256  512  256 64   128                                        __________________________________________________________________________

                  TABLE 4B                                                        ______________________________________                                        Activity of boronic acid derivatives against                                  bacterial cells when used by themselves.                                      MICs of inhibitors when used alone without amoxicillin,                       in μg/ml. The strains and plamids are the same as for Table                4A. The compounds were not tested above 128 μg/ml; here,                   "256" indicates no inhibition of cell growth.                                        BIPD DETHFB   9PHNB   3NPB  TH2B  TAZO                                 ______________________________________                                        EC       256    256      256   256   64    32                                 EC-T1    256    256      256   64    64    32                                 EC-T10   256    256      256   128   128   64                                 EC-AmpCEn                                                                              256    256      256   256   128   64                                 ECR-AmpCEc                                                                             128    256      256   128   128   64                                 ECR-     256    256      256   128   64    64                                 AmpCEnM                                                                       Ent-Der. 256    256      256   128   128   256                                ______________________________________                                    

                  TABLE 4C                                                        ______________________________________                                        Activity of TH2B against Pseudomonas aeruginosa in                            combination with ceftazidime.                                                                             MIC      MIC                                                                  cell culture                                                                           cell culture                                               β-lactamase                                                                        CAZ alone                                                                              CAZ/TH2B                                 Inhibitor                                                                            Organism   Expressed (μg/ml).sup.a                                                                       (μg/ml).sup.a                         ______________________________________                                        TH2B   Pseudomanas                                                                              AmpC      128      8/10*                                           aeruginosa (clinical                                                                     isolates)                                                   ______________________________________                                         .sup.a Broth dilution assays against the Pseudomonas aeruginosa clinical      isolates (from Hospital Ramon y Cajal in Madrid, Spain). The inhibitors       were used in combination with ceftazidime (CAZ). The concentration of CAZ     was varied by serial dilution, at a constant concentration of TH2B of 10      μg/ml. Dilution average of 11 clinical isolates. Range was 1/10            CAZ/TH2B to 64/10 CAZ/TH2B.                                              

Example 4

Testing of Compounds for Inhibition of β-Lactamases

Additional compounds were tested for inhibition of AmpC β-lactamase asdescribed in Example 2. The results are presented in Table 5 below. Thelast two compounds in Table 5 were synthesized by Key Organics,Cornwall, UK. The other compounds in Table 5 were obtained fromLancaster Synthesis, Windham, N.H., Aldrich Chemical, Milwaukee, Wis.,or Frontier Scientific, Logan, Utah.

                  TABLE 5                                                         ______________________________________                                         ##STR5##                                                                     R                       Ki AmpC (μM)                                       ______________________________________                                         ##STR6##               8.5 ± 1.8                                           ##STR7##               53.4 ± 6.1                                          ##STR8##               10.9 ± 0.6                                          ##STR9##               >>100                                                  ##STR10##              5.9 ± 0.3                                           ##STR11##              4.2 ± 1.1                                           ##STR12##              1.4 ± 0.1                                           ##STR13##              15.8 ± 0.8                                          ##STR14##              >>100                                                  ##STR15##              0.50 ± 0.05                                         ##STR16##              0.78 ± 0.08                                         ##STR17##              0.075                                                  ##STR18##              0.075                                                 ______________________________________                                    

Example 5

Antibacterial Activity

Two compounds (BZB and TH2B) were tested as described in Example 3against a wider range of bacteria using CAZ as the β-lactam antibiotic.All bacterial strains and plasmids are available from Jesus Blazquez andFernando Baquero, Servicio de Microbiologia, Hospital Ramon y Cajal,National Institute of Health, Madrid, Spain. The results are presentedin Table 6 below.

                  TABLE 6                                                         ______________________________________                                                         CAZ     BZB    CAZ-   CAZ-                                   Species/Enzyme Expressed                                                                       alone   only   BZB    TH2B                                   ______________________________________                                        MC4100/AmpC-Enter                                                                              32             1      4                                      MC4100/AmpC-E. coli                                                                             8             1      2                                      MC4100/AmpC-Enter (OmpR-)                                                                      32             1      4                                      MC4100/AmpC-E. coli (OmpR-)                                                                     8             2      2                                      MC4100/AmpC-Enter (OmpC-)                                                                      16             1      2                                      MC4100/AmpC-E. coli (OmpC-)                                                                    16             1      2                                      MC4100/AmpC-Enter (OmpF-)                                                                      32             1      4                                      MC4100/AmpC-E. coli (OmpF-)                                                                    32             1      2                                      Pseudomonas aeruginosa-1                                                                        8      512    8                                             (clinical isolate)                                                            Ps. aeruginosa-2 (clinical isolate)                                                            32      512    4                                             Ps. aeruginosa-3 (clinical isolate)                                                            64      512    4                                             Enterobacter cloacae derrepressed                                                              16      128    2                                             (clinical isolate)                                                            E. coli derrepr. (clinical isolate)                                                            16      128    2                                             Citrobacter freundii derrepressed                                                              16      128    2                                             (clinical isolate)                                                            ______________________________________                                         MC4100 is a strain of E. coli available from the American Type Culture        Collection, Rockyille, MD, accession number 35695.                            For AmpC plasmids, see Example 3.                                             OmpC and OmpF are porin channel proteins associated with the expression o     porin channels.                                                               OmpR is a regulatory protein that governs the expression of OmpF and OmpC     "-" indicates a mutant lacking one of these proteins that the wildtype        bacteria would ordinarily have.                                               The clinical isolates are from Hospital Ramon y Cajal in Madrid, Spain.  

We claim:
 1. A method of treating a β-lactam-antibiotic-resistantbacterial infection comprising administering to an animal suffering fromsuch an infectionan effective amount of a compound which is aβ-lactamase inhibitor, the compound having the formula: ##STR19##wherein: R is naphthalene, phenanthrene, or has one of the followingformulas: ##STR20## wherein ring system (2), (4), (5), (6), (7), (8),(9), (10), (13) or (14) is aromatic or nonaromatic;the atom center * is(R) or (S) in the case of chiral compounds; positions 1, 2, 3, 4, 5, 6,7 or 8 each independently is C, O or S; each R₁ independently is a lonepair, H, B(OH)₂, a halogen atom, CF₃, CH₂ CF₃, CCl₃, CH₂ CCl₃, CBr₃, CH₂CBr₃, NO₂, lower alkyl, CO₂ H, CHCHCOOH, CH₂ CH₂ CH₂ COOOH, SO₃ H, PO₃H, OSO₃ H, OPO₃ H, OH, NH₂, CONH₂, COCH₃, OCH₃, or phenyl boronic acid;or a pharmaceutically-acceptable salt thereof; and an effective amountof a β-lactam antibiotic.
 2. The method of claim 1 wherein R is (4). 3.The method of claim 2 wherein atom 1 is S or O and the remaining atoms2-6 are carbons.
 4. The method of claim 3 wherein the compound isbenzo[b]furan-2-boronic acid or benzo[b]thiophene-2-boronic acid.
 5. Themethod of claim 1 wherein R is (6).
 6. The method of claim 5 wherein thecompound is benzo[b]thiophene-3-boronic acid.
 7. The method of claim 1wherein R is (2).
 8. The method of claim 7 wherein atom 1 is S and theremaining atoms 2-4 are carbons or atom 2 is S or O and the remainingatoms 1 and 3-4 are carbons.
 9. The method of claim 8 wherein thecompound is thiophene-2-boronic acid, 3-formylthiophene-2-boronic acid,5-chlorothiophene-2-boronic acid, 4-methythiophene-2-boronic acid,5-acetylthiophene-2-boronic acid, or R-3-tetrahydrofuranylboronic acid.10. The method of claim 1 wherein the β-lactam antibiotic is amoxicillinor ceftazidime.
 11. A method of inhibiting a β-lactamase comprisingcontacting the β-lactamase with an effective amount of a compound havingthe formula: ##STR21## wherein: R is naphthalene, phenanthrene, or hasone of the following formulas: ##STR22## wherein: ring system (2), (4),(5), (6), (7), (8), (9), (10), (13) or (14) is aromatic ornonaromatic;the atom center * is (R) or (S) in the case of chiralcompounds; positions 1, 2, 3, 4, 5, 6, 7 or 8 each independently is C,N, O or S; each R₁ independently is a lone pair, H, B(OH)₂, a halogenatom, CF₃, CH₂ CF₃, CCl₃, CH₂ CCl₃, CBr₃, CH₂ CBr₃, NO₂, lower alkyl,CO₂ H, CHCHCOOH, CH₂ CH₂ C₂ CH₂ COOH, SO₃ H, PO₃ H, OSO₃ H, OPO₃ H, OH,NH₂, CONH₂, COCH₃, OCH₃, or phenyl boronic acid; or pharmaceuticallyacceptable salts thereof.
 12. The method of claim 11 wherein theβ-lactamase is produced by bacteria, and the bacteria are contacted withthe compound or salt thereof.
 13. The method of claim 11 wherein thecontacting takes place in vitro.
 14. A pharmaceutical compositioncomprising a compound which is a β-lactamase inhibitor having theformula: ##STR23## wherein: R is naphthalene, phenanthrene, or has oneof the following formulas: ##STR24## wherein: ring system (2), (3), (4),(5), (6), (7), (8), (9), (10), (13) or (14) is aromatic ornonaromatic;the atom center * is (R) or (S) in the case of chiralcompounds; positions 1, 2, 3, 4, 5, 6, 7 or 8 each independently is C, Oor S; each R₁ independently is a lone pair, H, B(OH)₂, a halogen atom,CF₃, CH₂ CF₃, CCl₃, CH₂ CCl₃, CBr₃, CH₂ CBr₃, NO₂, lower alkyl, CO₂ H,CHCHCOOH, CH₂ CH₂ CH₂ COOH, SO₃ H, PO₃ H, OSO₃ H, OPO₃ H, OH, NH₂,CONH₂, COCH₃, OCH₃, or phenyl boronic acid; orpharrnaceutically-acceptable salts thereof.
 15. The composition of claim14 wherein R is (4).
 16. The composition of claim 15 wherein atom 1 is Sor O and the remaining atoms 2-6 are carbons.
 17. The composition ofclaim 16 wherein the compound is benzo[b]furan-2-boronic acid orbenzo[b]thiophene-2-boronic acid.
 18. The composition of claim 14wherein R is (6).
 19. The composition of claim 18 wherein the compoundis or benzo[b]thiophene-3-boronic acid.
 20. The composition of claim 14wherein R is (2).
 21. The composition of claim 20 wherein atom 1 is Sand the remaining atoms 2-4 are carbons or atom 2 is S or O and theremaining atoms 1 and 3-4 are carbons.
 22. The composition of claim 21wherein the compound is thiophene-2-boronic acid,3-formylthiophene-2-boronic acid, 5-chlorothiophene-2-boronic acid,4-methylthiophene-2-boronic acid, 5-acetylthiophene-2-boronic acid, orR-3-tetrahydrofuranylboronic acid.
 23. The composition of claim 14further comprising a β-lactam antibiotic.
 24. The composition of claim23 wherein the β-lactam antibiotic is amoxicillin or ceftazidime.